Project Summary Membrane proteins comprise over 30% of the proteins encoded by the genome, and their efficient and accurate localization is essential for the structure and function of all cells. Compared to the well-studied co-translational protein targeting pathway, post- translational membrane protein targeting poses additional challenges due to the presence of highly hydrophobic transmembrane domains on the substrate protein. Our general goal is to use the Guided Entry of Tail-anchored protein (GET) pathway as a paradigm to understand the molecular mechanism of post-translational membrane protein targeting. Our specific goals are to understand the physical and functional coupling of the GET machinery with the cellular chaperone network, and to establish the roles of the latter in the conformational maintenance and triage of newly synthesized membrane proteins. In addition, we aim to decipher the molecular mechanisms by which targeting complexes containing tail anchored protein substrates are captured, remodeled and inserted by the receptor complex at the ER membrane. These studies will establish a comprehensive, high-resolution molecular model for this conserved and essential cellular pathway. Moreover, the lessons from the GET pathway will establish new roles and mechanisms for the cellular chaperone network, and suggest generalizable principles for ensuring the efficiency and fidelity of membrane protein localization and minimizing protein homeostasis stress during these processes. The proposed research is of a most basic nature, and will contribute profoundly to our general understanding of physiology and pathology of living cells at the molecular level. !